Diethylcarbamazine, TRP channels and Ca2+ signaling in cells of the Ascaris intestine

The nematode parasite intestine absorbs nutrients, is involved in innate immunity, can metabolize xenobiotics and as we show here, is also a site of action of the anthelmintic, diethylcarbamazine. Diethylcarbamazine (DEC) is used to treat lymphatic filariasis and activates TRP-2, GON-2 & CED-11 TRP channels in Brugia malayi muscle cells producing spastic paralysis. DEC also has stimulatory effects on ascarid nematode parasites. Using PCR techniques, we detected, in Ascaris suum intestine, message for: Asu-trp-2, Asu-gon-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9 and Asu-trpa-1. Comparison of amino-acid sequences of the TRP channels of B. malayi, and A. suum revealed noteworthy similarity, suggesting that the intestine of Ascaris will also be sensitive to DEC. We used Fluo-3AM as a Ca2+ indicator and observed characteristic unsteady time-dependent increases in the Ca2+ signal in the intestine in response to DEC. Application of La3+ and the TRP channel inhibitors, 2-APB or SKF 96365, inhibited DEC mediated increases in intracellular Ca2+. These observations are important because they emphasize that the nematode intestine, in addition to muscle, is a site of action of DEC as well as other anthelmintics. DEC may also enhance the Ca2+ toxicity effects of other anthelmintics acting on the intestine or, increase the effects of other anthelmintics that are metabolized and excreted by the nematode intestine.

Soil-transmitted helminths (STHs), including Ascaris, Trichuris, and hookworm, are a primary medical and public health concern in many developing countries. It is estimated that approximately 807 million to 1.2 billion people are infected with Ascaris lumbricoides 1 . Although not usually fatal, parasitic infections have a detrimental effect on morbidity, reducing worker productivity by 6.3 million Disability Adjusted Life Years (DALYs) per year. Parasitic infections affect worker health, output, and the performance of children in school. In livestock, infestations lead to reduced food yields, impacting economic returns and exacerbating poverty 2 .
Currently, there are no effective vaccines against STH infections, and treatment relies on the use of chemotherapeutics, usually using one of the three major classes of anthelmintics: benzimidazoles (albendazole/mebendazole), macrocyclic lactones (ivermectin), or nicotinic cholinergics (levamisole and pyrantel). There is a risk of resistance developing in humans associated with regular use of these compounds, as seen in animal parasites 3 . With the limited number of anthelmintic drugs and the risk of resistance development, identifying the modes of action of other existing anthelmintics and predicting rational anthelmintic combinations, which are more potent and effective, is anticipated to counter the resistance.
The classic drug diethylcarbamazine (DEC) is used to treat lymphatic filariasis caused by filarial parasites including Brugia malayi and Wucheria bancrofti 4,5 , but DEC has also been used to treat STH infections, including ascariasis 6 . Although DEC has been used for over seventy years, its mode of action is poorly understood. DEC's recently discovered actions are to open nematode muscle Transient Receptor Potential (TRP) channels, including the TRPMs, GON-2 & CED-11, and the TRPC, TRP-2 7 . The opening of muscle cell TRPs allows entry of Ca 2+ and transient spastic paralysis. TRP channels in nematodes are not only found in muscle cells, but they are also found distributed throughout the different tissues of nematodes including the intestine 8,9 . This suggests that DEC will also affect the cells of the intestine and impair its vital functions.
The nematode intestine is a long tube of single-layer columnar epithelial cells that connects the base of the pharynx to the anus. The intestine is vital for nematode survival. It is essential for digestion and the transport of ion solutes and nutrients [10][11][12][13][14] . It is involved in defense against environmental toxins and microbial infection by mediating innate immunity responses and the release of antimicrobial peptides 15,16 . The intestine is also a major site of metabolism of xenobiotic drugs by cytochrome P450 oxidative enzymes 17 : it excretes xenobiotic Results TRP channels in Ascaris. In the filarial nematode, Brugia malayi, DEC stimulates spastic muscle paralysis by opening muscle TRP channels of the TRPM group (Bma-GON-2 and Bma-CED-11) and the TRPC group (Bma-TRP-2) 7 . We blasted these genes against the A. suum (ASU) database (Wormbase) and found Asu-gon-2, Asu-ced-11, Asu-trp-2. We also found the TRPV genes, Asu-ocr-1 & Asu-osm-9, and the TRPA channel gene, Asu-trpa-1. We compared the sequences of these six TRP A. suum genes: Asu-gon-2, Asu-ced-11, Asu-trp-2, Asuocr-1, Asu-osm-9, and Asu-trpa-1 with the sequences of the same genes in: (1) the Clade III nematode parasites, Parascaris equorum (PEQ) and Brugia malayi (BMA); (2) the Clade V nematode, C. elegans (CEL); (3) the Clade I nematode parasite, Trichuris muris (TMU); (4) the schistosome, Schistosoma mansoni (SMA) and; (5) the fly, Drosophila melanogaster (DME). Accession number links to all sequences used can be found in Supplementary TableS3. The dendrogram, Fig. 1, shows that the TRP channels of A. suum and B. malayi are more closely related than to the TRP genes of C. elegans, S. mansoni, and D. melanogaster. The close relationship of the TRP channels of A. suum and B. malayi encouraged the hypothesis that TRP channels of A. suum and B. malayi have a similar pharmacology and sensitivity to DEC.

TRP channel expression in Ascaris intestine.
To determine if these TRP channel genes are expressed in the intestine and muscle bags of Ascaris, we generated screening primers targeting the coding region of these six genes (Supplementary Table S1). We compared the expression found in the intestine and expression found in the muscle bag regions of the same animal. We found that all six genes were expressed in both the intestine and the muscle bag regions ( Fig. 2; for original uncropped gel pictures see Supplementary Fig. S1) although, their band intensities varied between muscle and intestine. Thus, the TRP channels (Asu-GON-2, Asu-CED-11 & Asu-TRP-2) that are associated with mediating DEC effects on the muscle of the filarial parasite, Brugia malayi, are similar in sequence and present in the intestine of A. suum. TRP channels have tissue-specific expressions levels in Ascaris. Variations in band intensity for the different TRP channels between the intestine and muscle bag regions suggested differences in the relative mRNA expression levels for each channel. We used qPCR to measure the mRNA expression levels for each of the six TRP channels in the intestine relative to the reference gene, Asu-gapdh. We compared the TRP channel expression levels to paired muscle bags from the same Ascaris parasite.
Our analysis showed that Asu-gon-2 had a higher expression level in the intestine compared to the muscle bags (4.5-fold, Fig. 3, black bar). We observed that Asu-trp-2 had similar levels of expression in both the intestine and muscle bag (Fig. 3, blue bar). For Asu-ced-11, we observed a reduced expression level in the intestine compared to muscle, suggesting that the CED-11 function in the intestine is less significant, Fig. 3. For the TRPV channels, we observed that OCR-1 has a higher expression level in the intestine, (20-fold, Fig. 3, red bar), but that OSM-9 expression was relatively low in the intestine (Fig. 3). Lastly, for TRPA-1, qPCR showed relatively high levels of expression in the intestine (3.5-fold) compared to the muscle (Fig. 3, green bar). The different levels of expressions of the different TRP channel transcripts will relate to the different physiological functions of the A. suum cells of the intestine and cells of the muscles.
In B. malayi, DEC activates TRP channels that involve TRP-2, GON-2, and CED-11 subunits. The presence of clear and significant expression levels of Asu-gon-2 and similar expression of Asu-trp-2 but not Asu-ced-11 suggests that the GON-2 and TRP-2 channels are activated by DEC in the Ascaris intestine. We do not exclude possible effects of DEC on the TRPV channel, OCR-1, and the TRPA channel, TRPA-1, in the intestine of A. suum which have high levels of expression in the intestine. www.nature.com/scientificreports/ tion of the intestine preparation. In fresh preparations, it produced a rapid and smooth increase in the Fluo-3 fluorescence starting within 20 s of application. This Ca 2+ induced rise in cytosolic calcium declined immediately after the bath CaCl 2 concentration was returned to 1 mM. The time for the CaCl 2 signal to reach the peak was relatively fast, taking a mean of 3 min, Fig. 4A and D. We point out that we observed no major fluctuations in the Ca 2+ signal when the sample was constantly perfused long term with 1 mM CaCl 2 APF; there was only small spontaneous fluctuations that had an average amplitude of 0.6% (Supplementary Fig. S2A and B). Previous work on the muscle cells of the filarial nematode, B. malayi, showed that DEC opens their Transient Receptor Potential channels (TRPs), causing spastic paralysis 7 . We anticipated that DEC would also open TRP channels in the intestines of Ascaris. We bathed the intestines in 10 µM DEC for 5 min and observed that DEC produced a slow but uneven 'sometimes step-like' Ca 2+ signal in the intestine as opposed to the smoother rise caused by the CaCl 2 and was initiated within 30 s of the DEC application (Fig. 4B). The increase continued to rise after removal and washout of DEC before eventually declining; the Ca 2+ concentration reached its peak at a mean of 14.5 min after DEC exposure (Fig. 4D). The peak amplitude of the DEC signal was significantly smaller (10%) and slower than that produced by the application of 10 mM CaCl 2 (31%), Fig. 4C.

DEC stimulates a prolonged
Although the DEC calcium signal was reversible on washing, we used histology to check that the observed calcium signal was not associated with DEC induced cellular damage of the enterocytes. We exposed different Ascaris body flaps (intestine and underlying muscle cells) from the same worms to 10 µM DEC for 5 min and then washed them in 1 mM CaCl 2 APF for 10 min (average time to peak) or 60 min (average length of a recording). We then compared the enterocytes of the sections taken: before DEC treatment; 10 min after 5 min exposure to 10 µM DEC and; after the 60 min wash. We observed no evidence of DEC causing death or damage to the cell body of the enterocyte or their brush borders ( Supplementary Fig. S3). Based on these observations, DEC   www.nature.com/scientificreports/ generates a Ca 2+ signal increase with unique characteristics that is reversible on washing and, not associated with histological damage.

La 3+ inhibits DEC cytosolic Ca 2+ increases in normal extracellular Ca 2+ and inhibits DEC cytosolic Ca 2+ decreases in low extracellular Ca 2+ .
The TRP channels, GON-2/GTL-1 in the C. elegans intestine, have nearly identical pore domains [31][32][33][34] and exhibit high Ca 2+ selectivity, which is blocked rapidly by low extracellular La 3+ concentrations (100 µM) preventing movement of calcium through the plasma membrane into the enterocytes [34][35][36] . We tested the effects of 100 µM La 3+ on our DEC responses. Figure 5A shows a representative trace where 100 µM La 3+ blocks the DEC increase in Ca 2+ in normal extracellular Ca 2+ APF. Interestingly, in low extracellular calcium (no La 3+ ), DEC produces a reduction in the cytoplasmic calcium rather than an increase, due to leaching of intracellular calcium from the cell, Fig. 5B; and this leaching is inhibited by 100 µM La 3+ , Fig. 5C. These observations show that DEC can open Ca 2+ permeable plasma membrane channels in the enterocytes that are blocked by La 3+ without producing release of intracellular Ca 2+ . Figure 5D shows histograms of the significance and the means ± SEM changes in the Ca 2+ signal produced in the different experiments, n = 5 preparations for each experiment. The entry of Ca 2+ to, and loss of Ca 2+ from, the cytosol through the plasma membrane channels that is inhibited by a low-concentrations of La 3+ , is consistent with DEC opening TRP channels. The effects of La 3+ were readily washed off and control responses to our test 10 mM CaCl 2 then returned (not shown). Although La 3+ is recognized for blocking TRP channels of the nematode intestine, it is possible that other plasma membrane channels permeable to calcium could be a target of DEC. The plasma membrane channels may include the voltage-activated calcium Ca v channels, I CRAC and other calcium entry pathways that are also blocked by La 3+ . We used 2-APB and SKF 96365 to explore the pharmacology of the DEC calcium signaling response and the role of TRP channels in the response. 2+ . We used the broad-spectrum TRP channel antagonist, 2-APB (2-aminoethoxydiphenyl borate) which at low concentrations (10 µM) also has stimulatory effects of Store Operated Calcium entry (SOC) mediated by I CRAC channels 40 . We tested the effects of 10 µM 2-APB on DEC responses, Fig. 6. Application of 10 µM 2-APB alone had no effect on the Ca 2+ signal ( Fig. 6C; black bar). We found that 10 µM 2-APB did not inhibit the effect of 10 µM DEC if applied after the DEC: the Ca 2+ response continued in the presence of 2-APB and after removal of DEC, Fig. 6A. However, if 2-APB was applied at the same time as DEC, it prevented any increase in the intracellular Ca 2+ fluorescence ( Fig. 6B and C, white bar) until the 2-APB was removed, which then generated an increase in fluorescence that continued after DEC www.nature.com/scientificreports/ was removed ( Fig. 6B and C, light grey bar). The lack of 2-APB inhibition when applied after the DEC may be explained by calcium-induced calcium release into the cytoplasm once there is an initial rise in cytosolic calcium. We checked the response to 10 mM CaCl 2 after 2-APB and DEC applications to ensure that the preparation was still responding normally and saw the usual ~ 30% increase in the fluorescent signal (Fig. 6C, dark grey bar). Thus, 2-APB does not irreversibly inhibit the Ca 2+ signaling machinery. The histogram shown in Fig. 6C summarizes the mean % changes in Ca 2+ fluorescence changes from 6 similar experiments. There was no significant increase in Ca 2+ when 2-APB was co-applied with DEC (Fig. 6C, compare black and white bars), but when 2-APB was removed in the presence of DEC, we observed a significant increase in the Ca 2+ signal with its characteristic phenotype and similar peak ( Fig. 6B and C, compare white bar with the light grey bar). Although La 3+ can block other channels, including voltage-gated calcium channels, Ca v s, and other cation permeable plasma channels 37 , 2-APB has no inhibitory effect on voltage-gated calcium channels 38,39 and at low (10 µM) concentrations can stimulate SOC extracellular Ca 2+ entry pathways mediated by I CRAC (Orai) channels 40 . The inhibitory effects of La 3+ and 2-APB together can be explained by the effects of DEC opening TRP channels in the plasma membrane to promote Ca 2+ entry into the intestinal cells via TRP channels.

SKF 96365 inhibits DEC signaling.
We have pointed out that the broad-spectrum TRP inhibitor, 2-APB, may have off target effects including opening of I CRAC SOCs [38][39][40] and that La 3+ blocks calcium entry pathways including: TRPs and voltage-activated calcium Ca v channels 37 . We wanted to determine if our observed DEC www.nature.com/scientificreports/ signal included a role for a more specific TRP channel subtype. We exposed intestinal tissues to the TRPC selective antagonist SKF 96365 40,41 . We have previously demonstrated that 10 µM SKF 96365 is able to inhibit DEC mediated inward currents in Brugia muscles 7 . We exposed intestinal tissues to 10 µM SKF 96365 for 5 min and www.nature.com/scientificreports/ observed no changes in the Ca 2+ amplitude ( Fig. 6E and D, black bar). Upon application of 10 µM DEC in addition to SKF 96,365, we observed that the DEC mediated signal was inhibited, with the average amplitude being reduced to 3% ( Fig. 6E and D, white bar). The effect of SKF 96365 was washed off, reversing the inhibition, and allowing DEC to stimulate a Ca 2+ signal with an average amplitude of 11% ( Fig. 6E and D, light grey bar) and the response to the 10 mM Ca 2+ test to be unaltered (Fig. 6D,  Prolonged DEC exposure does not change TRP mRNA levels in the intestine or muscle. Maintained applications of diethylcarbamazine to adult B. malayi produces a temporary spastic paralysis that lasts for less than 4 h that is followed by desensitization and recovery 7 . We were interested in determining if there was accommodation to DEC in A. suum mediated by reducing TRP channel message during a 4-h exposure. We treated isolated intestine flaps and muscle flaps with 10 and 100 µM DEC for 4 h. Again, we used qPCR to measure relative mRNA levels of the six TRP channels in the treated flaps and compared them to untreated controls that were bathed in RPMI. We observed no significant changes in message levels for any TRP channels in either the intestines or muscle cells samples treated with either 10 µM or 100 µM DEC when using a one-way ANOVA with a post-hoc Tukey test ( Supplementary Fig. S5A and B). These observations show that the level of TRP channel transcript expression in the intestines and muscle cells of A. suum is not sensitive to DEC over this timescale.

Discussion
The nematode intestine carries out essential functions for survival, including: (1) digestion and nutrient absorption 10,12 ; (2) pH regulation via apical membranes V-type ATPases 18 ; (3) storage of lipids 43 ; (4) innate immunity 14,44 ; (5) secretion of bactericidal peptides 15 ; (6) drug metabolism with P450 cytochromes and UDP transferases 17 and; (7) excretion by organic-anion-transporter (OAT-1), P-glycoprotein-and MDR-transporters into the lumen of the intestine 14,18,45 . Damage to the cells of the intestine will adversely affect these vital functions. The intestine cells are columnar cells, electrically coupled to their neighbors, and polarized with the apical region bearing microvilli and the basolateral region facing the somatic body muscle cells. The digestion of food involves the secretion of proteases and lipases and the absorption of nutrients including glucose and lipids. It is facilitated by a range of selective transporters 46 . The processes of absorption of nutrients and secretion of proteases also require significant endocytosis and exocytosis utilizing different types of vacuoles and vesicles that are present in the intestinal cells; these vesicles are moved within the cell on an elaborate network of microtubules that includes a terminal web under the apical microvilli 11 . Absorption of Ca 2+ and Mg 2+ is mediated by TRPMlike ion-channels (GON-2) 35,36 .
The intestine of parasitic nematodes is increasingly being recognized as a major site of action of a number of anthelmintic drugs in addition to the neuromuscular system 47 . These anthelmintics now include:

Diethylcarbamazine (DEC):
The disturbance in the Ca 2+ homeostasis of the intestine by an action of DEC on TRP channels can disrupt vital functions of the enterocytes and will contribute to the anthelmintic action of DEC against ascariasis. DEC activates TRP-2, GON-2 & CED-11 channels that produce an inward sarcolemma current in Brugia malayi muscle cells where 2-APB and SKF 96365 are also effective as DEC antagonists 7 . The presence of TRP channel message in Ascaris intestine that is similar to that of B. malayi, Fig. 1 suggests that DEC will have the same effects on the A. suum intestine. The actions of La 3+ and 2-APB taken together on the intestine inhibiting the actions of DEC are explained by DEC also opening TRP channels in the intestine of A. suum. The effect of SKF 96365 also supports a role for TRP-2 channels. Fig. 7 shows a summary model of the action of DEC on the Ascaris intestine mediated by activating TRP-2, and GON-2 TRP channels leading to the entry of Ca 2+ into the intestinal cells. We have included here STIM-1 and ORAI-1 ( Supplementary Fig. S4 and Table S3) that is involved in storeoperated calcium entry and ryanodine receptors that are involved in calcium-induced calcium release, although details of their role in the DEC response requires further study. Message for GON-2 and TRP-2 channels are present in the A. suum intestine as they are in B. malayi muscle cells 7 . DEC also  www.nature.com/scientificreports/ produces excitability and depolarization in A. suum muscle cells 48 showing that DEC has actions on both muscle cells and intestine cells of related clade III nematode parasites, Fig. 1. We point out that we also found Asu-ced-11, ocr-1, osm-9 and Asu-trpa-1 message in the intestine and effects of DEC on these TRP channels were not excluded by our observations. However, the relatively high expression levels of Asu-gon-2 and the presence of Asu-trp-2 in the intestine compared with the muscle and their role in mediating DEC effects in B. malayi muscle favors GON-2 and TRP-2 as targets of the action for DEC in the intestine. Also, the TRP channel antagonists, 2-APB and SKF96365, inhibit the responses of the intestine to DEC as it does on B. malayi muscle, suggesting a similar mode of action. Further support for the action of DEC on TRP channels comes from the effects of La 3+ that inhibits TRP channels, and responses to DEC 35 and; interestingly, in the calcium-free conditions, DEC produces a fall in calcium, suggesting that cytosolic calcium is leached through the DEC activated TRP channels.

Benzimidazoles:
The intestine of nematodes is sensitive to the actions of the benzimidazole anthelmintics, mebendazole, and albendazole 19,49,50 . These drugs bind selectively to the β-tubulin of nematodes, disrupt microtubules, and the movement of digestive vacuoles and lysosomes, leading to autolysis of the intestinal cells 19,49 . 3. Levamisole: We have reported that the intestine expresses message for different acetylcholine gated ion channels subunits, including UNC-38, UNC-29, UNC-63, and ACR-8 21 . The application of the anthelmintic levamisole opens these nicotinic acetylcholine channels that produce a clear and characteristic calcium signal. The function of these channels in the intestine cells may be paracrine, to communicate locally with adjacent intestine cells and underlying muscle cells, because the cells are not electrically excitable like muscle cells or nerve cells. 4. The Bacillus thuringiensis toxin: Cry5B is a potent pore-forming toxin that can target multiple different species of gastrointestinal parasites including Ascaris suum [26][27][28] . Cry5B appears to have two modes of action: (1) binding selectively to the CDH-8 cadherin present on the surface of the cell membrane of C. elegans intestines, leading to oligomerization of the toxin and possible internalization 25 and; (2) interaction with glycolipids produced by BRE-5, a glycosyltransferase that catalyzes the addition of monosaccharides onto glycolipids, leading to Cry5B insertion into the membrane forming the pore 22  www.nature.com/scientificreports/ two anthelmintics 23 . Here, we have observed that DEC has a direct effect on the intestine, in addition to the known effects of levamisole 21 . In addition to levamisole, the two other cholinergic agonist anthelmintics, pyrantel and tribendimidine also have strong synergistic effects with Cry5B in C. elegans 23 that is also seen in the hookworm, Ancylostoma ceylanicum 24 . The observations that we describe here suggest a mechanism by which DEC will add to, and potentiate, the effects of albendazole, levamisole, ivermectin, and Cry5B in combinations treatments for STH infections.
We have pointed out above that the nematode parasite intestine is a site of action of DEC, the benzimidazoles, cholinergic anthelmintics like levamisole, and Cry5B. These anthelmintic drugs disrupt the normal Ca 2+ homeostasis and vital functions and suggest that combinations of these anthelmintics will have additive or synergistic effects. Adverse effects on the intestine will potentiate the effects of other anthelmintics that are metabolized or excreted by the intestine. One example is the macrocyclic lactone, ivermectin, which acts as positive allosteric modulators of the Glutamate-gated Chloride channel. P-glycoproteins have been suggested as candidate genes for ivermectin resistance by acting as efflux pumps. The P-glycoprotein gene pgp-9 has been repeatedly associated with macrocyclic lactone resistance in different nematode parasites 51,52 . Although P-glycoproteins are found in other nematode tissues, they are most strongly expressed in the intestine of the parasites 45,53 . The combination of albendazole with ivermectin or diethylcarbamazine is anticipated to enhance the efficacy of the treatment of STHs compared to albendazole alone, ivermectin alone, or diethylcarbamazine alone. This has, in fact, been seen in the study of the effects of albendazole, ivermectin, and diethylcarbamazine alone and combinations against ascariasis and trichuriasis 54 . The parasite intestine offers a common target where anthelmintics can have synergistic effects.

Materials and methods
Collection and maintenance of A. suum worms. Adult female A. suum worms were collected from the JBS Swift and Co. pork processing plant at Marshalltown, Iowa. Worms were maintained in Ascaris Ringers Solution (ARS: 13 mM NaCl, 9 mM CaCl 2 , 7 mM MgCl 2 , 12 mM C 4 H 11 NO 3 , Tris, 99 mM NaC 2 H 3 O 2 , 19 mM KCl and 5 mM glucose pH 7.8) at 32 °C for 24 h to allow for acclimatization before use in experiments. The solution was changed daily, and worms were used for a maximum of three days for experiments. All the worms were examined at the start of each day and excluded if they were damaged or immotile.

A. suum cDNA synthesis and RT-PCR detection of TRP channels. Dissection of muscle bags and
intestinal tissue was conducted on adult A. suum females as previously described, McHugh et al., 2020. Subsequently, muscle bags and intestinal tissue were homogenized separately in 1 ml of Trizol reagent using a mortar and pestle, followed by total RNA extraction according to the Trizol Reagent protocol (Life Technologies, USA). One microgram (1 µg) of total RNA from each tissue was used to generate cDNA by reverse transcription (RT) using SuperScript IV VILO™ Master Mix (Life Technologies, USA) following the manufacturer's protocol. PCR was conducted to detect the presence of Asu-gon-2, Asu-trp-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9, and Asu-trpa-1 using primers that were designed for targeting encoding regions of each gene (Supplementary Table S1). Asugapdh was used as a reference gene. Negative controls included enzyme, water, and both forward and reverse primers for the target with no cDNA template. The cycling conditions for PCR were an initial denaturation for 2 min at 95 °C, followed by 35 cycles of 95 °C for 30 s, 58 °C for 35 s, 72 °C for 45 s, and a final extension at 72 °C for 10 min using GoTaq® G2 Hot Start Green Master Mix (Promega, USA). The PCR products of each gene were then separated on an individual 1% Agarose containing SYBR® Safe DNA Gel Stain (ThermoFisher Scientific), for 45 min at 100 V, followed by visualization under UV light to confirm the presence of the genes. All photographs were acquired using Visionworks™ software (Analytik Jena) with an exposure setting of 3 s per 1 frame. Original gel pictures are presented in Supplementary Fig. S1.

Analysis of mRNA levels by quantitative real-time PCR.
To quantify the relative mRNA transcript levels of each identified TRP channel in the intestine and muscle bags, cDNA was synthesized from 1 µg of RNA from each adult female A. suum as described above. Target genes with fragments ranging from 150 to 200 bp were amplified by qPCR from each cDNA sample in triplicate. The same procedure was used for the reference gene Asu-gapdh. All primers for qPCR are presented in Supplementary Table S2. The quantitative PCR reaction mixture consisted of 1 μl of cDNA template, 1 μl of forward and reverse primer mix, and 10 μl of PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, ThermoFisher, USA), with the final volume made up to 20 μl with Nuclease-free water. The cycling conditions included an initial denaturation for 10 s at 95 °C, 40 cycles of 95 °C for 15 s and 58 °C for 30 s followed by a final melting curve step. Cycling was performed using a QuantS-tudio™ 3-96 well 0.1 mL Block Real time PCR Detection system (ThermoFisher, USA), and transcript quantities were derived by the system software, using the generated standard curves. mRNA expression levels for each subunit (Asu-gon-2, Asu-trp-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9, and Asu-trpa-1) were estimated relative to the reference gene (Asu-gapdh) using the Pfaffl Method. The qPCR experiments were repeated 3 times for each gene and for each DEC treatment (all subunit mRNA quantifications were performed in triplicate for each worm's muscle bag sample and intestinal tissue sample: 15 biological replicates each with three technical replicates).
Dendrogram construction. All protein sequences used for the construction of the dendrogram were acquired either from Wormbase Parasite, The European Nucleotide Archive (ENA) or UNIPROT repositories. Accession number links to sequences used for each gene of each organism can be found in Supplementary  Table 3. All protein alignments were done using MAFFT (http:// mafft. cbrc. jp/ align ment/ softw are/) 55 . Aminoacid similarity and identity was analyzed using MAFFT alignments. Percent identity was the percent ratio of www.nature.com/scientificreports/ identical amino-acids and the total number of residues in the longest sequence. Similarly, percent similarity was determined by dividing the number of identical and similar amino-acids by the total number of residues in the longest sequence (× 100). Phylogenetic analysis was done using the software MEGA X software 56 using the maximum likelihood method based on Le and Gascuel model 57 .
Preparation and loading Fluo-3AM. A  For all experiments, samples were exposed to solutions containing no added CaCl 2 and 100 µM LaCl 3 . All incubations were done in the absence of light to prevent degradation of the fluorescent dye. All solutions were delivered to the chamber under gravity feed through solenoid valves controlled using a VC-6 six-channel Valve Controller (Warner Instruments, Hamden, CT) through an inline heater set at 37 °C (Warner Instruments, Hamden, CT) at a rate of 1.5 mL/min. At the start of all experiments, intestinal preparations were left under blue light for a minimum of 3 min to promote settling and equilibration of the fluorescent signal and monitor for any spontaneous Ca 2+ signaling before application of any compound.
Measurement of Ca 2+ fluorescence. All recordings were performed on an Eclipse TE3000 microscope (20X/0.45 Nikon PlanFluor objective), fitted with a Photometrics Retiga R1 Camera (Surrey, BC, Canada). Light control was achieved using a Lambda 10-2 two-filter wheel system with a shutter controller (Lambda Instruments, Switzerland). Filter wheel one was set on a green filter between the microscope and camera. Filter wheel two was set on the blue filter between a Lambda LS Xenon bulb light box, which delivered light via a fiber optic cable to the microscope (Lambada Instruments, Switzerland). Blue light emission was controlled by using a shutter. Minimal illumination exposure was used to prevent photobleaching.
All Ca 2+ signal recordings were acquired and analyzed using MetaFluor 7.10.2 (MDS Analytical Technologies, Sunnyvale, CA) with exposure settings at 150 ms with 2 × binning. Maximal percent Ca 2+ signal amplitudes (ΔF) were calculated using the equation F1-F0/F0 × 100, where F1 is the fluorescent value and F0 is the baseline value. All F0 values were determined as being the value when any stimulus was applied to the sample for all traces analyzed.
Details of numbers of regions and numbers of preparations used for measurements of the Ca 2+ signals. Calcium signals from each intestine were collected from 50 square 50 µm × 50 µm areas across the intestine covering a total area of 125,000 µM 2 that included 800-1000 individual enterocytes. The average fluorescence amplitude was calculated for each intestinal exposure of all 50 regions. For the DEC experiments, intestines were exposed to 10 µM DEC for 5 min followed by 10 mM CaCl 2 as the positive control. For the 2-APB experiments intestines were exposed to 10 µM 2-APB for 5 min followed by 10 mM CaCl 2 . For the study of the effects of 2-APB on the DEC responses, preparations were exposed to 10 µM 2-APB and 10 µM DEC at the same time for 5 min before 2-APB was removed and DEC was left for an additional 5 min. 10 mM CaCl 2 was then used as a positive control. For SKF 96,365 experiments, 10 µM SKF96365 was applied for 5 min followed by applying of 10 µM SKF96365 + 10 µM DEC for 5 min. SKF96365 was removed, and the DEC was left on for an additional 5 min. 10 mM CaCl 2 was then used as a positive control. Finally, for the no added Ca 2+ and La 3+ experiments 10 µM DEC was applied for 5 min before subjected to 10 mM CaCl 2 .
Histology. Sections of Ascaris body sections were cut along the lateral line and opened, exposing the intestine. Sections were then incubated in 1 mM CaCl 2 APF containing 10 µM DEC for 5 min and then transferred to 1 mM CaCl 2 APF for 10 min, average time for Ca 2+ signal to reach peak (15 min total) or 55 min, the average length of recording (60 min total). Control samples were exposed to 1 mM CaCl 2 APF for 3 min.
Following treatment, the segments of adult Ascaris worms were preserved in 10% buffered neutral formalin. Preserved specimens were placed in histology cassettes for paraffin embedding and sectioning. Hematoxylin and eosin staining was performed on 5 µm slides as described previously 58 . Histological sections were reviewed by an ACVM board-certified parasitologist (Brewer). Sections were viewed and images were captured on an Olympus BX60 microscope fitted with an Olympus DP70 camera (Olympus, Tokyo, Japan). Image captures were achieved using Olympus DP controller software (Olympus, Tokyo, Japan) under 20 × magnification and exposure settings of 1/350 s per frame. www.nature.com/scientificreports/ Statistical analysis. Statistical analysis of all data was done using GraphPad Prism 5.0 (Graphpad Software, Inc., La Jolla, CA, USA). To ensure reproducibility, we repeated our experiments: the numbers of female worms, intestine preparations, the concentrations, and durations of applications of 2-APB, DEC, La 3+ are provided in the legends of the figures. Analysis of Ca 2+ amplitudes and time to peak were done using either unpaired or paired student t-tests P < 0.05. For qPCR experiments one-way analysis of variance (ANOVA), with a post-hoc Tukey test P < 0.05 was used. The data are presented as relative expression levels in mean ± SEM for each TRP channel subunit relative to Asu-gapdh in muscle bags and intestinal tissue. Comparisons were made using Tukey's post hoc test.
Chemicals. Source of chemicals: 2-APB and SKF96365 were procured from Tocris; Sigma Aldrich supplied all other chemicals.